Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference

Cancer Gene Therapy volume 17, pages 780–791 (2010)Cite this article

Subjects

Abstract

RNA interference (RNAi) is a natural cellular regulatory process that inhibits gene expression by transcriptional, post-transcriptional and translational mechanisms. Synthetic approaches that emulate this process (small interfering RNA (siRNA), short hairpin RNA (shRNA)) have been shown to be similarly effective in this regard. We developed a novel ‘bifunctional’ RNAi strategy, which further optimizes target gene knockdown outcome. A bifunctional construct (bi-sh-STMN1) was generated against Stathmin1, a critical tubulin modulator that is overexpressed in human cancers. The bifunctional construct is postulated to concurrently repress the translation of the target mRNA (cleavage-independent, mRNA sequestration and degradation) and degrade (through RNase H-like cleavage) post-transcriptional mRNA through cleavage-dependent activities. Bi-sh-STMN1 showed enhanced potency and durability in parallel comparisons with conventional shRNA and siRNAs targeting the same sequence. Enhanced STMN1 protein knockdown by bi-sh-STMN1 was accompanied by target site cleavage at the mRNA level showed by the rapid amplification of complementary DNA ends (RACE) assay. Bi-sh-STMN1 also showed knockdown kinetics at the mRNA level consistent with its multieffector silencing mechanisms. The bifunctional shRNA is a highly effective and advantageous approach mediating RNAi at concentrations significantly lower than conventional shRNA or siRNA. These results support further evaluations.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Irvine DV, Zaratiegui M, Tolia NH, Goto DB, Chitwood DH, Vaughn MW et al. Argonaute slicing is required for heterochromatic silencing and spreading. Science 2006; 313: 1134–1137.

    Article  CAS  PubMed  Google Scholar 

  2. Bailis JM, Forsburg SL . RNAi hushes heterochromatin. Genome Biol 2002; 3: Reviews1035.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Chu CY, Rana TM . Small RNAs: regulators and guardians of the genome. J Cell Physiol 2007; 213: 412–419.

    Article  CAS  PubMed  Google Scholar 

  4. Ghildiyal M, Zamore PD . Small silencing RNAs: an expanding universe. Nat Rev 2009; 10: 94–108.

    Article  CAS  Google Scholar 

  5. Grimm D, Kay MA . Therapeutic application of RNAi: is mRNA targeting finally ready for prime time? J Clin Invest 2007; 117: 3633–3641.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gewirtz AM . On future's doorstep: RNA interference and the pharmacopeia of tomorrow. J Clin Invest 2007; 117: 3612–3614.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Silva J, Chang K, Hannon GJ, Rivas FV . RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age. Oncogene 2004; 23: 8401–8409.

    Article  CAS  PubMed  Google Scholar 

  8. Wang S, Shi Z, Liu W, Jules J, Feng X . Development and validation of vectors containing multiple siRNA expression cassettes for maximizing the efficiency of gene silencing. BMC Biotechnol 2006; 6: 50.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Liu YP, Haasnoot J, ter Brake O, Berkhout B, Konstantinova P . Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron. Nucleic Acids Res 2008; 36: 2811–2824.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Li L, Lin X, Khvorova A, Fesik SW, Shen Y . Defining the optimal parameters for hairpin-based knockdown constructs. RNA (New York, NY) 2007; 13: 1765–1774.

    Article  CAS  Google Scholar 

  11. Pillai RS . MicroRNA function: multiple mechanisms for a tiny RNA? RNA (New York, NY) 2005; 11: 1753–1761.

    Article  CAS  Google Scholar 

  12. Zeng Y, Cai X, Cullen BR . Use of RNA polymerase II to transcribe artificial microRNAs. Methods Enzymol 2005; 392: 371–380.

    Article  CAS  PubMed  Google Scholar 

  13. Nemunaitis J, Senzer N, Khalil I, Shen Y, Kumar P, Tong A et al. Proof concept for clinical justification of network mapping for personalized cancer therapeutics. Cancer Gene Ther 2007; 14: 686–695.

    Article  CAS  PubMed  Google Scholar 

  14. Rana S, Maples PB, Senzer N, Nemunaitis J . Stathmin 1: a novel therapeutic target for anticancer activity. Expert Rev Anticancer Ther 2008; 8: 1461–1470.

    Article  CAS  PubMed  Google Scholar 

  15. Iancu C, Mistry SJ, Arkin S, Wallenstein S, Atweh GF . Effects of stathmin inhibition on the mitotic spindle. J Cell Sci 2001; 114 (Part 5): 909–916.

    CAS  PubMed  Google Scholar 

  16. Alli E, Yang JM, Hait WN . Silencing of stathmin induces tumor-suppressor function in breast cancer cell lines harboring mutant p53. Oncogene 2006; 26: 1003–1012.

    Article  PubMed  Google Scholar 

  17. Zhang HZ, Wang Y, Gao P, Lin F, Liu L, Yu B et al. Silencing stathmin gene expression by survivin promoter-driven siRNA vector to reverse malignant phenotype of tumor cells. Cancer Biol Ther 2006; 5: 1457–1461.

    Article  CAS  PubMed  Google Scholar 

  18. Mistry SJ, Atweh GF . Therapeutic interactions between stathmin inhibition and chemotherapeutic agents in prostate cancer. Mol Cancer Ther 2006; 5: 3248–3257.

    Article  CAS  PubMed  Google Scholar 

  19. Ngo TT, Peng T, Liang XJ, Akeju O, Pastorino S, Zhang W et al. The 1p-encoded protein stathmin and resistance of malignant gliomas to nitrosoureas. J Natl Cancer Inst 2007; 99: 639–652.

    Article  CAS  PubMed  Google Scholar 

  20. Wang R, Dong K, Lin F, Wang X, Gao P, Wei SH et al. Inhibiting proliferation and enhancing chemosensitivity to taxanes in osteosarcoma cells by RNA interference-mediated downregulation of stathmin expression. Mol Med (Cambridge, MA) 2007; 13: 567–575.

    Article  CAS  Google Scholar 

  21. Azuma-Mukai A, Oguri H, Mituyama T, Qian ZR, Asai K, Siomi H et al. Characterization of endogenous human argonautes and their miRNA partners in RNA silencing. Proc Natl Acad Sci USA 2008; 105: 7964–7969.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J 2004; 23: 4051–4060.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Lagos-Quintana M, Rauhut R, Meyer J, Borkhardt A, Tuschl T . New microRNAs from mouse and human. RNA (New York, NY) 2003; 9: 175–179.

    Article  CAS  Google Scholar 

  24. Mendell JT . miRiad roles for the miR-17-92 cluster in development and disease. Cell 2008; 133: 217–222.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wu L, Fan J, Belasco JG . Importance of translation and nonnucleolytic ago proteins for on-target RNA interference. Curr Biol 2008; 18: 1327–1332.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD . Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 2005; 123: 607–620.

    Article  CAS  PubMed  Google Scholar 

  27. Leuschner PJ, Ameres SL, Kueng S, Martinez J . Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep 2006; 7: 314–320.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Pizzorno MC, O'Hare P, Sha L, LaFemina RL, Hayward GS . Trans-activation and autoregulation of gene expression by the immediate-early region 2 gene products of human cytomegalovirus. J Virol 1988; 62: 1167–1179.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R, Donoghue M et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 2004; 432: 173–178.

    Article  CAS  PubMed  Google Scholar 

  30. Roush SF, Slack FJ . Micromanagement: a role for microRNAs in mRNA stability. ACS Chem Biol 2006; 1: 132–134.

    Article  CAS  PubMed  Google Scholar 

  31. Wu L, Fan J, Belasco JG . MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci USA 2006; 103: 4034–4039.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Zeng Y, Cullen BR . Sequence requirements for micro RNA processing and function in human cells. RNA (New York, NY) 2003; 9: 112–123.

    Article  CAS  Google Scholar 

  33. Silva JM, Li MZ, Chang K, Ge W, Golding MC, Rickles RJ et al. Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 2005; 37: 1281–1288.

    Article  CAS  PubMed  Google Scholar 

  34. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005; 33: e179.

    Article  PubMed  PubMed Central  Google Scholar 

  35. McManus MT, Petersen CP, Haines BB, Chen J, Sharp PA . Gene silencing using micro-RNA designed hairpins. RNA (New York, NY) 2002; 8: 842–850.

    Article  CAS  Google Scholar 

  36. Landthaler M, Gaidatzis D, Rothballer A, Chen PY, Soll SJ, Dinic L et al. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA (New York, NY) 2008; 14: 2580–2596.

    Article  CAS  Google Scholar 

  37. Robb GB, Brown KM, Khurana J, Rana TM . Specific and potent RNAi in the nucleus of human cells. Nat Struct Mol Biol 2005; 12: 133–137.

    Article  CAS  PubMed  Google Scholar 

  38. Langlois MA, Boniface C, Wang G, Alluin J, Salvaterra PM, Puymirat J et al. Cytoplasmic and nuclear retained DMPK mRNAs are targets for RNA interference in myotonic dystrophy cells. J Biol Chem 2005; 280: 16949–16954.

    Article  CAS  PubMed  Google Scholar 

  39. Jarve A, Muller J, Kim IH, Rohr K, MacLean C, Fricker G et al. Surveillance of siRNA integrity by FRET imaging. Nucleic Acids Res 2007; 35: e124.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Chiu YL, Rana TM . siRNA function in RNAi: a chemical modification analysis. RNA (New York, NY) 2003; 9: 1034–1048.

    Article  CAS  Google Scholar 

  41. Rossi JJ . Expression strategies for short hairpin RNA interference triggers. Human Gene Ther 2008; 19: 313–317.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge the generous support of the Baylor Health Care System Foundation—Lane Newsom Drennan Fund, the Jasper L and Jack Denton Wilson Foundation, the WW Caruth, Jr Foundation Fund of Communities Foundation of Texas, Merkle Family Foundation and the Mary Crowley Cancer Research Centers. We would also like to acknowledge Brenda Marr and Susan Mill for their competent and knowledgeable assistance in the preparation of this paper.

Author information

Authors and Affiliations

  1. Gradalis, Inc., Dallas, TX, USA

    D D Rao, P B Maples, N Senzer, P Kumar, Z Wang, B O Pappen, Y Yu, C Haddock, C Jay, A P Phadke, S Chen, A Tong, D Shanahan & J Nemunaitis

  2. Mary Crowley Cancer Research Centers, Dallas, TX, USA

    N Senzer, D Shanahan & J Nemunaitis

  3. Texas Oncology PA, Dallas, TX, USA

    N Senzer & J Nemunaitis

  4. Baylor Sammons Cancer Center, Dallas, TX, USA

    N Senzer & J Nemunaitis

  5. General and Oncology Surgery Associates, Dallas, TX, USA

    J Kuhn

  6. Van Andel Research Institute, Grand, Rapids, MI, USA

    D Dylewski, S Scott, D Monsma & C Webb

Authors
  1. D D Rao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P B Maples
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. N Senzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Z Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. B O Pappen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Y Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. C Haddock
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. C Jay
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. A P Phadke
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. S Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. J Kuhn
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. D Dylewski
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. S Scott
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. D Monsma
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. C Webb
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. A Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. D Shanahan
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. J Nemunaitis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J Nemunaitis.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rao, D., Maples, P., Senzer, N. et al. Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference. Cancer Gene Ther 17, 780–791 (2010). https://doi.org/10.1038/cgt.2010.35

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/cgt.2010.35

Keywords

This article is cited by

Search

Advanced search

Quick links